Blood platelets play an essential role in hemostasis. The purpose of this proposal is to investigate the cytoskeletal mechanics of platelet formation using a mouse megakaryocyte (MK) culture system. Platelet formation follows a defined set of morphogenetic shape changes driven by microtubules (MTs) and actin filaments within the MK cytoskeleton. It begins with the extension of large pseudopodia from the MK that appear to use cortical bundles of MTs to elongate into proplatelets which ultimately form prominent coils of MTs at their bulbous ends. Linear arrays of MTs that line the proplatelet shaft serve as tracks for the translocation of platelet components into developing platelets. Repeated bending and branching of proplatelets plays a crucial role in platelet formation. This actin-dependent process, which amplifies proplatelet ends, is essential for generating the beaded appearance of proplatelets. This MK culture system will be used to investigate the role of MTs and actin filaments in the mechanics of platelet formation. Aim I will define how MT forces elongate proplatelet processes and determine how the marginal MT coil forms during platelet morphogenesis. Dynamic changes in the MT cytoskeleton will be observed as it functions in living MKs to establish the contribution of MT assembly and/or sliding in proplatelet elongation and MT coil formation. In Aim 2, structural and biochemical methods will be used to characterize and define the mechanism of organelle and granule transport along MTs into developing platelets. Aim 3 will examine the mechanism of actin-dependent proplatelet bending/branching. In the last aim, we will examine platelet morphogenesis at the molecular level and define when the membrane skeleton of the discoid platelet assembles and is locked into place by the vWf receptor-filamin-actin linkage. Understanding the cytoskeletal mechanics of platelet formation may provide insights into how platelets are produced and yield strategies for treatment of thrombocytopenia.